1. Field of the Invention
This invention generally relates to a novel antibody-dextran-phycobiliprotein conjugate. In particular the present invention relates to a new antibody-aminodextranphycoerythrin conjugate, a method of making said conjugate, and a method for detecting biological substances in an assay using said conjugate.
2. Description of the Prior Art
There has always been a need to develop systems that can accurately and reliably detect and quantitate relatively low levels of biological substances in an assay. The demand for such systems has resulted in the development of a variety of detection methods in which the number of probe molecules per targeted site of interest is amplified, thus making it considerably easier to detect target sites.
For example, enhanced light absorption is used in enzyme immune assays in which substrate absorption is enhanced by large substrate turnovers and use of the high affinity avidin-biotin system See A. M. Shamsuddin and C. C. Harris, Arch. Pathol. Lab. Med. 107, 514-517 (1983); Adler-Storthz, et al., J. Clin. Microbiol. 18, 1329-1334 (1983); and J. A. Madri and K. W. Barwick, Lab. Invest. 48, 98-107 (1983). Enhanced light scatter is obtained in the side scatter from polystyrene beads coated with colloidal gold particles as described in U.S. Pat. No. 5,552,086, issued Sep. 3, 1996 to Siiman et al. Enhanced light emission is used in indirect fluorescence staining of cell receptor sites with multiple layers of phycoerythrin-streptavidin attached to biotinylated antibody at cell receptor sites as described by J. H. M. Cohen et al., J. Immunol. Methods 99, 53-58 (1987) and H. Zola et al., J Immunol. Methods, 135, 247-255 (1990). Enhanced light-induced photochemistry is used in excitation of chlorin e.sub.6 coupled through dextran to anti-T-cell monoclonal antibody to enhance singlet oxygen production as described by A. R. Oseroff et al., Proc. Natl. Acad. Sci., USA 83, 8744-8748 (1986). The single, common feature in the above methods is the increase in the number of probe molecules or particles per targeted site.
Increasing the number of probe molecules or particles per targeted site, however, does not always work. For example, H. M. Shapiro describes one attempt at amplification of fluorescence signals by Tomas Hirshfeld et al., at Block Engineering, wherein several hundred fluorescein molecules were attached to a synthetic polymer, polyethylenimine, which was then conjugated with antibody. The method did not work because fluorescence emission from fluorescein molecules was quenched due to the short nearest neighbor distances between fluorophores on the same polymer molecule. See PRACTICAL FLOW CYTOMETRY, 3rd edition, H. M. Shapiro, Wiley-Liss, New York, N.Y., 1995, p. 277.
Fluorescent dextrans have been used for fluorescence amplification, and numerous fluorescent dextrans are commercially available. See HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 6th edition, R. P. Haugland, Molecular Probes, Inc., Eugene, Oreg. 97402, 1996. Fluorescent dextrans consist of soluble dextrans (that is, 10,000, 40,000, 70,000, 500,000, and 2,000,000 daltons) conjugated with various fluorescent dyes such as fluorescein, dansyl, rhodamine, and Texas Red. The degrees of substitution in these fluorescent dextrans are 1-2 dye molecules per dextran of 10,000 daltons, 2-4 dye molecules per dextran of 40,000 daltons, 3-6 dye molecules per dextran of 70,000 daltons, about 64 dye molecules per dextran of 500,000 daltons, and about 134 dye molecules per dextran of 2,000,000 daltons. Higher degrees of substitution than these usually lead to quenching and non-specific interactions. Conjugated dextrans are also available as so-called "lysine-fixable", that is, they have incorporated lysine residues which can be used for further reaction, such as covalent attachment of antibody molecules. Fluorescein isothiocyanate (FITC) derivatives of dextran and poly-L-lysine with degrees of substitution ranging from 0.003 to 0.020 molecules of FITC per molecule of glucose and from 0.003 to 0.01 molecule of FITC per molecule of lysyl residue, are commercially available from sources, such as Sigma Chemical Company. For the largest molecular weight dextran listed (2,000,000 daltons) up to 33 to 222 molecules of FITC per molecule of dextran are available; and for a 70,000 dalton poly-L-lysine molecule, up to 5.5 molecules of FITC are available.
Aminodextran of sufficiently large molecular weight can accommodate multiple antibody molecules. The linearity of the polymeric sugar chain of antibody-dextran complexes is an advantage in providing access to targeted antigenic sites on cells without incurring steric hindrance that might occur with a globular polymeric molecule as a carrier. U.S. Pat. No. 5,527,713 issued Jun. 18, 1996, to Bolton et. al., describes the conjugation of anti-CD3 monoclonal antibody to aminodextran (1X-aminodextran, .about.1,000,000 daltons, 7% diamine substitution; 5X-aminodextran, .about.350,000 daltons, 20% diamine substitution) under saturating conditions of antibody on dextran to give a T3 antibody:aminodextran molar ratio in the conjugates of 37:1 for T3-1X-Amdex and 20:1 for T3-5X-Amdex, thus showing that aminodextran can be effectively loaded with many large protein molecules.
Another attempt at fluorescence amplification uses the fluorescent dye rhodamine. See Shechter et al., Proc. Natl. Acad. Sci., USA 75, 2135-2139 (1978). Higher than usual fluorescence intensities were obtained for the peptide hormones insulin and epidermal growth factor, by covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted with rhodamine molecules (i.e., 7:1). This was accomplished while still retaining some binding affinity of the hormone for its receptor (which is one of the basic requirements of any process of this kind).
Another currently available family of fluorescent dyes is the phycobiliproteins. The phycobiliproteins are a family of macromolecules found in red algae and blue-green algae. Each phycobiliprotein molecule contains a large number of chromophores. An antibody molecule directly labeled with fluorescein will have between 1 and 3 chromophores associated with it. An antibody molecule directly labeled by conjugation with a phycobiliprotein may have as many as 34 associated chromophores, each with an absorbance and quantum yield roughly comparable to those of fluorescein. Thus, phycoerythrin, (PE), a member of the phycobiliprotein family, is among the brightest fluorescent dyes currently available. Conjugated to an antibody, PE has been used to detect interleukin-4 in a fluorescent plate assay and found in M. C. Custer and M. T. Lotze, J. Immunol. Methods, 128, 109-117(1990), to be the only tested fluorophore that produced adequate signal.
In PE there is a monodisperse population of fluorescent groups that are already embedded in a protein. PE exhibits maximal absorbance and fluorescence without susceptibility to either internal or external fluorescence quenching so that attachment of two or more PE molecules to a polymeric carrier should not quench PE fluorescence. The net fluorescence intensity from a PE-polymer complex should be the sum of fluorescence intensities from individual PE molecules. Heretofore, it has not been possible to directly conjugate more than a single PE molecule (MW, 240,000 daltons) to an IgG antibody (MW, 160,000 daltons) without destroying or adversely affecting antibody activity. See U.S. Pat. No. 4,520,110, issued May 28, 1985 to Stryer et al., and U.S. Pat. No. 4,859,582, issued Aug. 22, 1989 to Stryer et al.
On a molar basis, one molecule of PE has a fluorescence yield that is equivalent to at least 30 fluorescein or 100 rhodamine molecules at comparable wavelengths. Thus, one would expect that the largest molecular weight FITC-dextran conjugate with the greatest degree of FITC substitution would have the potential to yield a fluorescence enhancement factor of 222/30=7.4 over a single PE molecule. However, with so many FITC units per dextran carrier and a broad distribution in the molecular weight of each dextran, it is difficult to synthetically mimic the monodisperse population of fluorescent groups that occurs naturally in molecules of PE. The spread in fluorescent intensities from one molecule of FITC-dextran complex to another offsets the large enhancement factor that may be numerically anticipated. With respect to rhodamine, the 7:1 rhodamine-to-carrier molar ratio falls far short of the 100:1 rhodamine to PE ratio needed for equivalent fluorescence intensity.
Therefore, no enhancement factor greater than one, relative to PE fluorescence, to date has been reported for the above FITC derivatives of dextran or poly-L-lysine. This is so, even though other fluorescent dyes are available and one might expect an enhancement factor of 7.4 over a single PE molecule. Thus, there is still a need for the amplification of fluorescence emission signals which uses a direct fluorescent marker to achieve emission intensities greater than those of about 1:1 antibody-PE conjugates.